Research Project 2 focuses on investigating the unique features of populations of tumor cells, both in various regions of primary and metastatic tumor, as well as in the bloodstream. The First Aim involves extensive specimen sampling of a small number of patients, including various regions of the primary tumor such as the leading invasive edge, the tumor epicenter (among others), as well as tumor cells within afferent and efferent blood vessels of the tumor and tumor cells in blood vessels over time. These population sets of cells will be characterized according to features related to metastability, including overall cytomorphologic structure, surface and cytoplasmic evidence of loss of epithelial-ness, live/dead nuclear features, and E- to N-cadherin switching. The resulting data will provide a tumor topology across the human organism and over time, yielding insight into which populations of tumor cells are important in the process of metastasis. The Second Aim focuses on the third microenvironment, the bloodstream, which is a transient, yet critical environment for cells undergoing hematogenous metastasis. Investigations are directed at the architecture of tumor cell travel groups in the blood, associ ations between CTCs and other nucleated cells in the bloodstrea m including immunologically active cells, scavenger cells (monocytes), and coating cells (platelets) that may play a critical role in CTC survival/destruction, interactions with non-cellular protein components such as those of the coagulation cascade, and m arkers associated with natural killer cell activity. Methodologies include the incorporation of newly developed techniques to study CTCs in human cancer patient blood samples, utilizing fluorescent immunocytochemistry and standard morphologic stains, combined with sophisticated digital imaging strategies and software applications. The Third Aim will use the data collected in the first two aims along with clinical patient correlation data to robustly simulate the behaviors of cancer cells as they leave the primary tumor and travel through the blood to metastatic sites.